RNA degradation Flashcards by Joe Bentley

What are kinetic proofreading processes coupled to?

Ribosome assembly and pre-mRNA assembly

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What 5’ protection does mRNA have?

mG7 cap has a Cap Binding Complex (CBC)
Triphosphorylated 5’ inhibits the Rai1 and Xrn2 exonucleases

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What 3’ protection does mRNA have?

PolyA tail with a PABP to inhibit 3’ exonuclease activity
Histone mRNA has a stem loop with binding proteins instead of polyA

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What does RNA surveillance do?

Dampens ncRNA expression so it doesn’t affect gene expression and genome stability
If RNA isn’t processes, RNA decay is the default pathway
Toxic RNA repeats accumulate in the brain of ALS patients

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How are the proteosome and exosome similar?

Degrade protein and RNA respectively
Both 4 tiered structures with a central channel
β subunits degrade subunits as they have chymotrypsin/trypsin/caspase activity

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What are TNTs?

Terminal nucleotide transferers (TNT) provides an oligo (A/U) tail for RNase to bind to in the exosome

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How does RNA helicase Ski2 aid the exosome?

Unwinds RNA recondary structures and stem loops so ribonucleases can bind and degrade them

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How do CUTs (Cryptic unannotated transcripts) degrade RNA?

Nrd1 and Nab3 are recruited to CUTs and interact with RNAPII and RNA
TRAMP polyadenylates CUT to target it to the exosome
Sen1 binds Nab3 and terminates mRNA synthesis
Mtr4 and Ski2 (RNA helicases) have arch domains that bind adaptors on RNA

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How do the adaptors on RNA determine what is degraded via the CUT system?

Adaptors can allow MTR4 complex to degrade polyA+
Adaptors can allow NEXT complex to degrade polyA-

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What are Xrn1 and Xrn2?

5’-3’ exonuclease. Acts on 5’ monophasphates
Xrn1- cytoplasmic
Xrn2- nuclear
Xrn2 functions in RNA processing, surveillance and transcription termination.

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How are aberrantly capped RNAs dealt with?

Rai1 has pyrophosphohydrolase activity which converts triphosphate to monophosphate
NAD caps are removed by Rai1
Dxo1 decaps nonmethylated caps and has 5’ exonuclease activity

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What does Rai1 do?

Has pyrophosphohydrolase activity which converts triphosphate to monophosphate.
Removes NAD caps

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What does Dxo1 do?

decaps nonmethylated caps and has 5’ exonuclease activity

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How are nonsense mutations dealt with in the ribosome?

Upf1 has RNA helicase activity, binds eRF3 and kinetic proofreads of RNA
This can downregulate genes from upstream ORFs and 3’ UTRs
Degrades products of alternative splicing, so crucial to gene expression

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Describe Tsp-mediated degradation?

tmRNA (truncated mRNA) in a ribosome is degraded by the 3’ exoribonuclease RNase R
Tsp protease recognises the degron tag on the polypeptide on the ribosome

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Describe translation termination

What a stop codon is reached, release factors Rf1 and Rf2 hydrolyse the polypeptide from the P site
RF3 is a GTPase which allows RF1 and 2 to be released
Lots of other factors dissociate from the ribosome

How does trans-translation degrade RNA without a stop codon?

tmRNA on the A-site functions as an Ala-tRNA. When the sORF on tmRNA is translated, it has a C-terminal tag that marks it to Tsp degradation

How are mRNAs without stop codons degraded?

The Ski7 CTD binds to a ribosomal A site, recruiting the exosome and Ski complex
Ski2 helicase binds to the small ribosomal subunit and threads mRNA through the exit site to the exosome

What can cause ribosome stalling?

Damaged nucleotides
Structural elements
Rare codons
Lots of basic residues

How can mRNA be degraded after ribosome stalling?

No-go decay
Non-stop decay for truncated mRNA (Ski2 and 7)

What happens after two ribosomes collide?

Hel2 (ubiquitin ligase) ubiquitinates the S3 and S10 ribosomal subunits which recruits mRNA degredation and quality control complexes
Rqc2 adds CAT tails and Ltn1 ubiquitinates elongating polypeptides
Cue2 cleaves mRNA from ribosome A-site

Describe translation initiation

40S ribosomal subunit binds to Met-tRNAi and mRNA to form an initiation complex
40S subunit scans 5’ UTR of mRNA to find start codon
eIF2-GTP -> eIF2-GDP causes 48S initiation complex to form
60S subunit associates when eIF5b is hydrolysed

Describe translation elongation

Amino-tRNAs are inserted into the polypeptide chain, causing ribosome to translocate along mRNA

How is ATF4 transcription affected by no stress?

High eIF2-GTP around the gene
Ribosome interacts with uORF1 and initiates at uORF2
ATF4 is not transcribed

How is ATF4 transcription affected by stress?

High eIF2-P and less ternary complex Ribosome bypasses inhibitory uORF2 and the ternary complex is initiated at the ATF4 gene, allowing its expression

What are uORFs used for?

mG7-cap dependent translation Used for non-canonical translation initiation Controls downstream expression positively or negatively

What is the rate-limiting step of translation

eIF2-GTP hydrolysis which recruits the ternary complex

What are internal ribosomal entry sites (IRES)?

Enable viruses to hijack host cell translational machinery Enables cap-independent translation initiation Used under global translational down-regulation

What does ribosome profiling show?

How much is translated What is translated Different aspects of translation Defects

How can ribosome profiling of ORFs be read?

Sharp 5' and 3' boundaries for start/stop sites Frame of translation is determined by the p-site

How is ribosome profiling done?

1. Translation is inhibited to halt ribosomes 2. Polysomes are isolated from the cell extract via centrifugation 3. RNA not protected by the ribosome is degraded with RNases 4. Ribosomes and their 28-32nt RNA fragments are isolated via density gradient centrifugation Sequencing and library prepararion

What are some strengths of ribosome profiling?

No need for genetic manipulation Quantifiable Sensitive (identifies rare translation events) Shows precise locations of genes Detects changes in protein expression

What are some limitations of ribosome profiling?

Ribosomes have to be frozen Artifacts from antibiotics to halt translation Short RNA fragements can lead to ambiguity to identify similar sequences Assumes elongation rate is the same Assumes each ribosome associated with RNA completes translation

What are sORFS?

small ORFs encode proteins with no known function Found in 5' UTR and overlap with exons Found in 3' UTR Found intergenically (between genes) Has codon periodicity Sensitive to inhibitors and translation

What is GTIseq?

Translation is inhibited with antibiotics Ribosomes are then left at the start site, so identifies these, event uORFs with non-AUG start sites

What does piwi-intercting RNA do?

Silences transposons in germ line cells and stem cells to maintain genetic stability Strong promotors which disrupt neighbouring genes piRNA signal is increased via ping-pong amplification Degrade transposons

What causes pontocerebellar hypoplasia?

Mutations in exosome core subunits EXOSC3 and 8 Disruption of tRNA/snRNA causes ncRNAs to accumulate

What are the symptoms of pontocerebellar hypoplasia?

Cerebellar atrophy Spinal motor neuron degeneration